CN108883412B - Microfluidic device for detecting target genes - Google Patents
Microfluidic device for detecting target genes Download PDFInfo
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- CN108883412B CN108883412B CN201780019041.7A CN201780019041A CN108883412B CN 108883412 B CN108883412 B CN 108883412B CN 201780019041 A CN201780019041 A CN 201780019041A CN 108883412 B CN108883412 B CN 108883412B
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Abstract
The present invention relates to a microfluidic device for detecting a target gene, comprising: a plurality of capillary flow tubes, wherein one side of the plurality of capillary flow tubes is immersed in a sample container containing a sample solution to flow the sample solution by a capillary phenomenon; and a microbead filler section disposed on a flow path of the sample solution inside one side of each of the capillary flow tubes, wherein each of the microbead filler sections includes: a filler conduit disposed on the microchannel so as to form a part of a flow path of the sample solution; a plurality of microbeads received in close contact with each other in the filler conduit such that voids are formed between the microbeads; and a probe linker formed on a surface of each of the microbeads, wherein the probe linker is amplified via complementary binding to a target gene in a sample solution to detect the target gene. The microfluidic device of the present invention can significantly reduce detection time.
Description
Technical Field
The present invention relates to a microfluidic device for detecting a target gene, and in particular, to a microfluidic device for detecting a target gene, which uses a phenomenon of amplifying a target gene so as to block a gap formed by microbeads or reduce the size of the gap, to detect various pathogenic viruses through target gene amplification.
Background
Efficient amplification of a target nucleic acid is an important factor for detection of nucleic acid, DNA sequencing, cloning, and the like. There are various methods for amplifying nucleic acids, such as Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), self-Sustained Sequence Replication (SSR), nucleic acid sequence dependent amplification (NASBA), and Strand Displacement Amplification (SDA).
Many of the above methods have the following disadvantages: the accuracy of the quantitative measurements of the method is relatively low and requires expensive equipment, and the disadvantages are particularly more and more severe when more than one target needs to be analyzed simultaneously.
In order to compensate for such disadvantages, isothermal amplification (isothermal amplification) has been invented, and in particular, Rolling Circle Amplification (RCA) methods have received much attention. That is, several techniques such as PCR have been used so far for detecting DNA, but the methods are disadvantageous because they are time-consuming, inefficient and require high cost and high manpower.
The PCR method comprises the following steps: denaturing in which the DNA is separated into single strands by heating the DNA in a reaction solution containing a primer pair, a template, a polymerase, and dntps; annealing, wherein primers complementary to each isolated DNA strand are ligated to the template by reducing the temperature; and polymerization, in which a new strand is polymerized by increasing the temperature through a polymerization reaction using a polymerase, and this amplification exponentially increases the DNA strand in proportion. However, the PCR process should go through the above steps and thus inevitably induce a temperature change. Therefore, the apparatus for PCR must have a temperature controller and a heating means. If PCR is used for amplification of a target nucleic acid in a lab-on-a-chip (LOC) or the like, a temperature controller and a heater for PCR reaction are further required in addition to a detection device for LOC, disadvantageously resulting in a complicated device and an increased cost for the device.
In order to solve the above disadvantages, several isothermal amplification methods have been proposed. Loop-mediated isothermal amplification (LAMP) is one of the isothermal amplification methods and uses 6 amplification primers to generate a multi-loop product with branching. Such LAMP is not suitable for early diagnosis or biosensor because of the use of primary Reverse Transcriptase (RT) to detect target RNA.
The RCA method is proposed as another isothermal amplification method. Advantageously, the RCA method does not require the temperature change necessary for PCR amplification as described above, and thus it is possible to amplify the target nucleic acid in an isothermal state. Therefore, it is possible to amplify without a temperature controller in a process requiring amplification, thereby reducing the complexity and cost of the apparatus.
In the Linear Rolling Circle Amplification (LRCA) method, a target NDA sequence is hybridized (Hybridizing) with an open loop probe to form a complex and then an amplified target loop is generated by Ligation (Ligation), and thereafter a primer sequence and a DNA polymerase are introduced. The amplification target loop forms a template in which new DNA is formed, and an extension is made from a primer that extends into a contiguous sequence of repetitive sequences complementary to the amplification target loop, thereby generating thousands of copies of nucleic acid per hour.
As a method for further development, an exponential RCA (exogenous RCA; ERCA) method was developed. In the ERCA method, additional primer sequences that bind to the replication sequence complementary to the amplification target loop are used to provide a new amplification center, thereby exponentially increasing amplification proportionally. In the ERCA method, the strand displacement (strand displacement) method is continued, but is limited to a method in which the initial single-stranded RCA product is used as a template for another DNA synthesis by using an isolated single-stranded primer attached to the product without additional RCA.
Another method using Molecular Padlock Probes (MPPs) and Rolling Circle Amplification (RCA) is disclosed (C.Larsson et al, Nature methods 2004, 1, 227). This method has several advantages and allows for the amplification of complementary nucleic acids in circular MPPs through the identification of target nucleic acid sequences, as well as high specificity. In particular, sensitivity is increased by direct binding of RCA products without further purification. The RCA reaction can be initiated on the surface by immobilizing the target nucleic acid probe to the surface of a material such as gold, quartz, or the like through a simple chemical surface treatment.
Meanwhile, a paper "DhITACT: DNA hydrogel Formation by Isothermal Amplification of Complementary targets in Fluidic Channels (17.6.2015, Advanced Materials, Volume 27, phase 23, 3513-3517) (DhITACT: DNAhydro gel Formation by Isothermal Amplification of Complementary Target in Fluidic Channels (June 17, 2015, Advanced Materials, Volume 27, Issue 23, Pages 3513-3517)' "discloses a technique in which an RCA reaction surface is provided on the bottom of a microchannel followed by two hours of reaction with a sample solution and single-stranded extension of DNA and self-assembly in the form of multiple dumbbells occurs, whereby the DNA starts to form a hydrogel shape and flow in the corresponding channel is excluded.
However, the technique disclosed in lihayan paper has a disadvantage in that it takes more than two hours to perform the test since the RCA reaction surface should be formed on the bottom surface of the microchannel, and then the tester must wait until the entire microchannel is blocked by amplification at the RCA reaction surface.
Disclosure of Invention
Technical problem
Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a microfluidic device for detecting a target gene, which can significantly reduce a detection time, in which the target gene is detected by a phenomenon of blocking or reducing the size of voids generated by microbeads through amplification of the target gene in order to detect various pathogen viruses by detecting the gene.
It is another object of the present invention to provide a microfluidic device for detecting a target gene, in which it is possible to quantitatively analyze the target gene according to detection based on various methods.
Technical solution
According to the present invention, the above object is achieved by a microfluidic device for detecting a target gene, comprising: a plurality of capillaries partially submerged in a sample container containing a sample solution, and in which the sample solution flows by a capillary phenomenon, and a bead filler disposed at one portion in each capillary, the one portion being disposed on a flow path of the sample solution, wherein each of the bead fillers includes: a filler tube disposed at the capillary tube so as to partially constitute a flow path of the sample solution; a plurality of microbeads contained in the filler tube and in close contact with each other to form gaps between the microbeads; and a probe linker formed on a surface of each of the microbeads, wherein the probe linker is configured to amplify a target gene in a sample solution by complementarily binding to the target gene, thereby detecting the target gene.
Here, the initial volume of the sample solution contained in the sample container may be set such that the sample solution reaches the bead filler through a capillary by a capillary phenomenon, wherein the gap may be blocked or the size of the gap may be reduced by amplifying a target gene induced by complementary binding between the target gene in the sample solution reaching the bead filler and the probe linker, and wherein when the sample solution is further introduced into the sample container, the target gene may be detected based on whether the sample solution flows to at least one of an opposite side of the bead filler or a final travel distance of the sample solution.
In addition, the probe linker of each bead filler may be configured to detect a different target gene.
Meanwhile, according to another embodiment of the present invention, the above object may be achieved by a microfluidic device for detecting a target gene, comprising: a sample chamber containing a sample solution; a microchannel connected to the sample chamber and through which a sample solution flows; and a bead filler disposed on a flow path of the sample solution in the microchannel; wherein the microbead filler comprises: a filler tube disposed at the microchannel so as to partially constitute a flow path of the sample solution; a plurality of microbeads contained in the filler tube and in close contact with each other to form gaps between the microbeads; and a probe linker formed on a surface of each of the microbeads, wherein the probe linker is configured to amplify a target gene in a sample solution by complementarily binding to the target gene, thereby detecting the target gene.
Here, the gap may be blocked or reduced in size by amplification of the target gene induced via complementary binding between the target gene and the probe linker, thereby changing a final travel distance of the sample solution, a time taken for the final travel distance, and a flow rate of the sample solution, and wherein the target gene is detected by one of the final travel distance, the time taken for the final travel distance, and the flow rate.
In addition, the microfluidic device may further include a negative pressure chamber disposed on an opposite side of the sample chamber to be in fluid communication with the microchannel, the negative pressure chamber applying a negative pressure from the outside to flow the sample solution through the microchannel, wherein the gap may be blocked or the size of the gap may be reduced by amplification of the target gene induced via complementary binding between the target gene and the probe linker, thereby detecting the target gene according to a change in the negative pressure applied by the negative pressure chamber.
In addition, the diameter of the microbeads may be set to have a size ranging from 0.1 to 100 micrometers according to the type of the target gene so that the target gene can pass through the gap.
Further, the microbead filler may include meshes respectively disposed at both ends of the filler tube to prevent loss of the microbeads.
Further, the sample chamber, the microchannel, and the bead filler may be provided in plural forms to be respectively arranged in parallel, and the bead contained in one of the bead fillers has no probe linker.
In addition, the sample chamber, the microchannel, and the bead filler may be provided in plural form to be respectively arranged in parallel, and the probe linker of each bead filler is configured to detect different target genes.
Further, the microchannel may include: a first flow channel connected to the sample chamber; and a plurality of second flow channels branched from the first flow channels, wherein a bead filler may be provided in plural form to be disposed in each of the second flow channels, wherein beads contained in each bead filler may have different diameters.
Further, the probe adaptor may include a coating portion coated on a surface of the microbead, a primer attached to the coating portion, and a template complementarily bound to the primer, wherein the template may include a first binding portion complementarily bound to the target gene, a second binding portion complementarily bound to the primer, and a third binding portion complementarily bound in the template to form a dumbbell, and wherein the first binding portion is divided and formed at both ends of the template, and the second binding portion is formed between the divided third binding portions.
Further, the coating portion may comprise one or more selected from the group consisting of: 5-hydroxydopamine hydrochlorate, norepinephrine, epinephrine, pyrogallol, DOPA (DOPA; 3, 4-Dihydroxyphenylalanine (3, 4-Dihydroxyphenylalanine)), catechin, tannic acid, pyrogallol, catechol, heparin catechol, polyglucose catechol, poly (ethylene glycol, catechin), poly (ethyleneimine, catechol), poly (methyl methacrylate) catechol, hyaluronic acid catechol, polylysine catechol, and polylysine.
Further, the primer may comprise one or more selected from the group consisting of: thiol (thio), amine (amine), hydroxyl (hydroxyl), carboxyl (carboxyl), isothiocyanate (isothiocyanate), NHS ester, aldehyde (aldehyde), epoxide (epoxide), Carbonate (Carbonate), HOBt ester, glutaraldehyde (glutamide), carbamate (carbamate), imidazole carbamate (imidazole carbamate), maleimide (maleimide), aziridine (azidine), sulfone (sulfolene), vinyl sulfone (vinylsulfone), hydrazine (hydrazine), phenyl azide (phenyl azide), benzophenone (benzophenone), anthraquinone (anthraquinone), and Diene (Diene) groups, and wherein the ends of the primers are modified.
Advantageous effects
With the above arrangement, in order to detect various pathogenic viruses by detecting genes, the target genes are detected by a phenomenon of blocking or reducing the size of voids generated by microbeads through amplification of the target genes, thereby making it possible to provide a microfluidic device for detecting the target genes that can significantly reduce the detection time.
Furthermore, it is possible to quantitatively analyze a target gene based on detection based on various methods.
Drawings
Fig. 1 is a view showing a microfluidic device for detecting a target gene according to one embodiment of the present invention.
Fig. 2(a) and 2(b) are views illustrating a method of detecting a target gene using a microfluidic device for detecting a target gene according to an embodiment.
Fig. 3 is a view showing a microfluidic device for detecting a target gene according to one embodiment of the present invention.
Fig. 4 is a view illustrating a bead filler of a microfluidic device for detecting a target gene according to an embodiment of the present invention.
Fig. 5 to 7 show a microfluidic device for detecting a target gene according to still another embodiment of the present invention.
[ description of reference numerals ]
1. 100, 100a, 100b, 100 c: a microfluidic device;
3: a sample container;
5: a sample solution;
10: a capillary tube;
110: a sample chamber;
120. 120a, 120b, 120c, 120d, 120 e: a negative pressure chamber;
130: a microchannel;
131: a first flow channel;
132a, 132b, 132c, 132d, 132 e: a second flow path;
30. 140, 140a, 140b, 140c, 140d, 140 e: filling of microbeads;
32. 141: microbeads;
142: a mesh;
33. 143: a probe linker;
144: a void;
31. 145: a filler tube;
151: a stirring rod;
152: and a magnet.
Detailed Description
According to the present invention, the present invention relates to a microfluidic device for detecting a target gene, and the microfluidic device comprises: a plurality of capillaries partially submerged in a sample container containing a sample solution, and in which the sample solution flows by a capillary phenomenon, and a bead filler disposed at one portion in each capillary, the one portion being disposed on a flow path of the sample solution, wherein each of the bead fillers includes: a filler tube disposed at the capillary tube so as to partially constitute a flow path of the sample solution; a plurality of microbeads contained in the filler tube and in close contact with each other to form gaps between the microbeads; and a probe linker formed on a surface of each of the microbeads, wherein the probe linker is configured to amplify a target gene in a sample solution by complementarily binding to the target gene, thereby detecting the target gene.
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Fig. 1 is a view showing a microfluidic device 1 for detecting a target gene according to one embodiment of the present invention. Referring to fig. 1, a microfluidic device 1 for detecting a target gene according to one embodiment of the present invention includes a plurality of capillaries 10 and a bead filler 30.
The plurality of capillaries 10 are configured to have a size such that the sample solution 5 can flow into the capillaries by a capillary phenomenon. In the embodiment, for example, as illustrated in fig. 1, six capillaries are arranged adjacent to each other, but the arrangement of the tubes is not limited thereto.
The plurality of capillaries 10 are configured such that one end portion of the tube, i.e., the lower portion of the tube as shown in fig. 1, is immersed in the sample container 3 containing the sample solution 5, and the sample solution 5 flows into the tube through an inlet at the lower side of the tube and flows upward by a capillary phenomenon.
A bead filler 30 is disposed in each capillary 10 on the flow path of the sample solution 5. Here, for example, the microbead filler 30 is manufactured separately from the microfluidic device 1 and then inserted into the capillary 10 when the microfluidic device is manufactured.
Meanwhile, the bead filler 30 according to the embodiment may include a filler tube 31, a plurality of beads 32, and a probe linker 33, as shown in fig. 1.
The filler pipe 31 is arranged on the flow path of the sample solution 5, i.e., in the capillary 10, to constitute some of the flow path of the sample solution 5. Further, the plurality of microbeads 32 contained in the filler tube 31 are in close and compact contact with each other, and there are gaps between the microbeads that are in close and compact contact with each other.
Here, the diameter of the microbeads 32 depends on the type of target gene and the size of the voids varies according to the diameter of the target gene. In this regard, the size of the void is set such that the target gene passes through the void and can be adjusted by adjusting the size of the microbead 32. In the embodiment, for example, the diameter of the microbeads 32 is determined to be in the range of 0.1 to 100 micrometers depending on the type of target gene.
Further, the bead filler 30 according to the embodiment may include meshes 142 respectively disposed at both ends of the filler tube 31 (see the embodiment in fig. 4). Advantageously, the inner diameter of the mesh 142 has a size that does not allow release of the microbeads 32 and does not interfere with the flow of the sample solution 5.
Meanwhile, a probe linker 33 is formed on the surface of each microbead 32. Further, the probe linker 33 is configured so as to perform amplification by complementary binding to the target gene in the sample solution 5, thereby detecting the target gene.
In an embodiment, probe connectors 33 are included in the paper "DhITACT: the formation of a DNA hydrogel by isothermal amplification of complementary targets in a fluidic channel (17.6.2015, advanced materials, vol.27, 23, p.3513-3517). That is, for example, the probe linker 33 includes a coating portion, a primer, and a template.
The coating portion is coated on the surface of the microbead 32 and formed of a material to which the primer is attached and fixed. As described in the above paper, examples of coating portions may include one or more selected from the following group: 5-hydroxydopamine hydrochlorate, norepinephrine, epinephrine, pyrogallol, DOPA (DOPA; 3, 4-Dihydroxyphenylalanine (3, 4-Dihydroxyphenylalanine)), catechin, tannic acid, pyrogallol, catechol, heparin catechol, polyglucose catechol, poly (ethylene glycol) catechol, poly (ethyleneimine) catechol, poly (methyl methacrylate) catechol, hyaluronic acid catechol, polylysine catechol, and polylysine (polylysine).
The primer is immobilized at the coating portion and the template is complementarily bound to the primer. Here, the template may include a first binding portion complementarily binding to the target gene, a second binding portion complementarily binding to the primer, and a third binding portion complementary in the template to form a dumbbell shape. Further, the first coupling portions are divided and formed at both ends of the template, and the second coupling portions are formed between the divided third coupling portions.
Here, examples of the primer may include one or more selected from the group consisting of: thiol (thio), amine (amine), hydroxyl (hydroxyl), carboxyl (carboxyl), isothiocyanate (isothiocyanate), NHS ester, aldehyde (aldehyde), epoxide (epoxide), Carbonate (Carbonate), HOBt ester, Glutaraldehyde (glutamide), carbamate (carbamate), imidazole carbamate (imidazole carbamate), maleimide (maleimide), aziridine (azidine), sulfone (sulfolane), vinyl sulfone (vinylsulfone), hydrazine (hydrazine), phenyl azide (phenyl azide), benzophenone (benzophenone), anthraquinone (anthraquinonone), and Diene (Diene) groups, wherein the terminal ends are modified.
With the above arrangement, the target gene is bound to the probe linker 33 of the example and amplified, and the amplified target gene forms a hydrogel. Since it has been disclosed in the above papers, a detailed explanation will be omitted.
As described above, the hydrogel is generated by amplification of complementary binding of the target gene to the probe linker 33 formed on the surface of each microbead 32, thereby blocking the gap between the microbeads 32 and reducing the size of the gap. The clogging of the voids and the reduction in the size of the voids will become resistance and in turn prevent the sample solution 5 from rising or decreasing the speed of rising due to the capillary phenomenon, thereby detecting the target gene.
Referring to fig. 2(a) and 2(b), a method of detecting a target gene using the microfluidic device 1 according to the above embodiment will be described.
First, as shown in fig. 2(a), a sample solution 5 is poured into the sample container 3. Here, the initial amount (WL1) of the sample solution 5 injected into the sample container 3 is set such that the sample solution 5 reaches the microbead filler 30 through the capillary 10 by a capillary phenomenon (see CL 1).
Here, the target gene in the sample solution 5 passed through the bead filler 30 is amplified by complementarily binding the target gene to the probe linker 33 of the bead filler 30, and then the target gene becomes hydrogel, thereby blocking the gap between the beads 32 and reducing the size of the gap.
Here, if the probe linker 33 of each bead filler 30 is provided to detect a different target gene, only the gap of the bead filler 30 having the corresponding target gene will be blocked and only the size of the gap of the bead filler 30 having the corresponding target gene will be reduced.
Next, as shown in fig. 2(a) and 2(b), when the sample solution 5 is further supplied into the sample container 3 (see WL2), the sample solution 5 should flow upward via the capillary 10. Here, if the sample solution 5 cannot flow to the opposite side of the bead filler 30 by amplification of the target gene, the presence of the corresponding target gene can be recognized.
Referring to fig. 2(b), in the two capillaries 10 on the left, it can be seen that the sample solution 5 passes through the bead filler 30 and rises (see CL2a), and this shows that the sample solution 5 does not have a target gene bound to the probe linker 33 of the bead filler 30.
In contrast, in the two capillaries 10 on the right, it can be seen that the sample solution 5 does not pass through the bead filler 30 because the bead filler 30 is clogged, and this shows that the sample solution 5 has the target gene bound to the probe linker 33 of the bead filler 30. And, in the middle capillary, it can be seen that the sample solution 5 has a relatively small amount of the target gene since the void is not completely blocked or it takes time to block the void.
In the above examples, it was tested whether the voids were blocked by adding the sample solution. In an alternative method, the capillary 10 is submerged in the sample solution, and then after a certain time, for example after a time sufficient for the target gene to be amplified, the capillary 10 is submerged deeper in the sample solution. Thus, if the sample solution 5 flowing upward through the capillary 10 cannot flow to the opposite side of the bead filler 30 due to amplification of the target gene, it is possible to recognize the presence of the target gene.
Here, in order to facilitate visual recognition as to whether the sample solution has moved to the upper portion of the capillary 10, the color of the sample solution may be controlled. For example, paper that changes color when wet is arranged at an upper portion of the microbead filler 30 in the capillary 10. With this arrangement, if the paper located in the capillary 10 changes color when the paper is wetted with the sample solution, it is recognized that the target gene is not present in the capillary 10.
Hereinafter, referring to fig. 3 and 4, a microfluidic device 100 for detecting a target gene according to another embodiment will be described in detail.
Referring to fig. 3 and 4, a microfluidic device 100 for detecting a target gene (hereinafter, referred to as "microfluidic device 100") according to another embodiment may include a sample chamber 110, a microchannel 130, and a bead filler 140. Furthermore, the microfluidic device 100 according to an embodiment may include a negative pressure chamber 120.
The sample chamber 110 is arranged at one side of the microfluidic device 100 and contains a sample solution. The microchannel 130 is in fluid communication with the sample chamber 110, and the sample solution contained in the sample chamber 110 flows through the microchannel 130.
The bead filler 140 is disposed on a flow path of the sample solution. Here, the bead filler is manufactured separately from the microfluidic device 100 and then installed in the microchannel 130 when the microfluidic device 100 is manufactured. For example, when an upper substrate having the sample chamber 110, the micro channel 130, and the fluid pressure chamber is attached to a transparent base substrate in the up-down direction in order to manufacture the microfluidic device 100, the upper substrate is attached to the base substrate in a state in which the bead filler 140 is inserted into the micro channel 130 of the upper substrate.
Meanwhile, as shown in fig. 4, the bead filler 140 according to an embodiment of the present invention may include a filler tube 145, a plurality of beads 141, and a probe linker 143. Here, the bead filler 140 corresponds to the bead filler of the embodiment in fig. 1 and a detailed explanation of the bead filler 140 is omitted. As shown in fig. 4, the bead filler 140 according to the embodiment may include meshes 142 respectively disposed at both ends of the filler tube 145 to prevent the beads 141 from being lost. Advantageously, the inner diameter of the mesh 142 is sized to prevent the microbeads 141 from releasing and not interfering with the flow of the sample solution.
Here, the hydrogel is generated by amplification of complementary binding of the target gene to the probe linkers 143 formed on the surface of each microbead 141, thereby blocking the gaps 144 between the microbeads 141 and reducing the size of the gaps 144, thereby causing a change in the final travel distance of the sample solution flowing through the microchannel 130, the time taken to reach the final travel distance, and the flow rate of the sample. Further, it is possible to detect the target gene by using at least one of the final travel distance, the travel time, and the flow rate.
Referring back to fig. 3, the negative pressure chamber 120 is disposed on an opposite side of the sample chamber 110 and is in fluid communication with the microchannel 130. A negative pressure from the outside is applied by the negative pressure chamber 120 to flow the sample solution through the microchannel 130.
Here, if the gap 144 is blocked or the size of the gap 144 is reduced by amplification due to complementary binding of the target gene to the probe linker 143, the pressure that has been continuously applied by the negative pressure chamber 120 will be changed, and the presence of the target gene can be recognized.
According to the above embodiment, a plurality of microbeads 141 constitute the microbead filler 140, and probe linkers 143 formed on the microbeads 141 are bound to target genes. Then, the target gene is amplified, and the hydrogel generates and blocks the voids 144 or reduces the size of the voids 144 during amplification, thereby causing changes in the final travel distance, travel time, flow rate, and/or pressure, the detection of which allows the presence of the target gene to be identified.
Furthermore, testing can be performed by blocking or plugging the voids 144 formed by the microbeads 141, rather than blocking the entire microchannel 130 as disclosed herein, thereby making it possible to significantly reduce testing time. Furthermore, the reaction area may be increased by forming the probe linker 143 on the surface of each microbead 141, instead of forming the reaction area only on the bottom surface as disclosed in the above paper, thereby making it possible to significantly reduce the test time.
Further, the presence of the target gene can be detected by the clogging phenomenon of the gap, and the target gene can also be quantitatively evaluated by using the change in the final travel distance, the travel time, the flow rate, or the pressure, or the like. For example, if there is a large number of target genes, the chance of reaction increases and then the gap 144 is blocked more quickly, and eventually the distance traveled decreases, which can be quantified based on statistical methods, thereby enabling quantitative assessment of the target genes.
Fig. 5 shows a microfluidic device 100a according to a further embodiment of the present invention. As shown in fig. 5, a microfluidic device 100a according to yet another embodiment may include a plurality of sample chambers 110, a plurality of microchannels 130, and a plurality of bead fillers 140. Here, the sample chamber 110 and the micro channel 130 are connected to each other one-to-one to form a flow path of the sample solution and a plurality of flow paths are arranged in parallel. In fig. 5, for example, two sample chambers 110 and two microchannels 130 are arranged in parallel to form two flow paths. A bead filler 140 is disposed in each microchannel 130, respectively.
Here, the bead 141 contained in one of the bead fillers 140 does not have the probe linker 143. Referring to fig. 5, as described above, for example, the bead 141 of one of the bead fillers 140 has a probe linker 143 and the bead 141 of the other of the bead fillers 140 does not have the probe linker 143.
In an example, each sample chamber 110 contains the same sample solution therein, and then the sample solution flows. Here, if the bead filler 140 has a probe linker, the binding and amplification of the target gene as described above causes the bead filler to block or the size of the gap 144 to decrease, thereby restricting the flow of the sample solution. In contrast, if the bead filler 140 does not have the probe linkers 143, the sample solution flowing through the bead filler 140 flows without restriction.
Further, if the bead filler 140 has the probe-linkers 143, a final travel distance that the sample solution having passed through the bead filler 140 has traveled before the blocking of the bead filler 140 stops the sample solution is measured, and a travel time of the sample solution is measured, and then the final travel distance and time of the sample solution is compared with those of the bead filler 140 without the probe-linkers 143, whereby the initial concentration of the target gene can be quantified.
Here, in the embodiment of fig. 5, for example, each flow path is provided with a negative pressure chamber 120. Further, it is of course noted that one end of each micro channel 130 is connected to one negative pressure chamber 120, and the sample solution is flowed by applying a negative pressure from one negative pressure chamber 120.
In addition, in the embodiment of fig. 5, the microbeads 141 of each of the microbead packings 140 may have different probe linkers 143, respectively, so as to bind to different target genes. That is, since each probe linker 143 binds to a different target gene to amplify the target gene, it is possible to simultaneously detect a plurality of target genes by using one microfluidic device 100.
Fig. 6(a) and 6(b) show a microfluidic device 100b according to yet another embodiment of the present invention. The embodiment as shown in fig. 6(a) and 6(b) is a modification of the embodiment of fig. 5 and, for example, each sample chamber 110 is provided with a stir bar 151.
Here, the stirring rod 151 is configured to be rotated by rotation of a magnet 152 disposed outside the microfluidic device 100. The sample solution contained in the sample chamber 110 is stirred by the stirring rod, and then the target gene in the sample solution is widely dispersed, thereby facilitating binding and amplification in the bead filler 140.
Fig. 7 shows a microfluidic device 100c according to a further embodiment of the present invention. Referring to fig. 7, a microchannel 130 of a microfluidic device 100c according to still another embodiment of the present invention may include a first flow channel 131 connected to a sample chamber 110, and the first flow channel 131 is branched into a plurality of second flow channels 132a, 132b, 132c, 132d, 132 e. Further, bead fillers 140a, 140b, 140c, 140d, 140e are disposed in the second flow channels 132a, 132b, 132c, 132d, 132e, respectively.
Here, the microbeads 141 contained in each of the microbead fillers 140a, 140b, 140c, 140d, 140e may have different inner diameters. The probe linkers 143 formed in each of the microbeads 141 may be configured to bind to the same target gene.
In the above arrangement, even when the concentration of the target gene in the sample solution is low, the bead fillers 140a, 140b, 140c, 140d, 140e having the beads 141 with a smaller diameter may be clogged. The clogging of the bead fillers 140a, 140b, 140c, 140d, 140e depends on the concentration of the target gene in the sample solution, and in turn, the clogging of the bead fillers occurs according to the order of the diameter size of the beads 141. Therefore, the target gene in the sample solution can be quantitatively evaluated based on the diameter of the microbead 141 that is finally generated as a blockage.
Here, in the embodiment of fig. 7, for example, a plurality of second flow passages 132a, 132b, 132c, 132d, 132e are connected to each of the negative pressure chambers 120a, 120b, 120c, 120d, 120e, respectively. Alternatively, it should be noted that the ends of the second flow channels 132a, 132b, 132c, 132d, 132e are merged to be connected to one negative pressure chamber.
Although several embodiments of the present invention have been shown and described above, it is obvious that those skilled in the technical idea of the present invention can easily design the embodiments within the scope of the technical idea or spirit included in the description of the present invention. The scope of the invention is to be determined by the appended claims and their equivalents.
Industrial applicability
The invention is applied to the field of detecting various pathogen viruses by detecting genes.
Claims (13)
1. A microfluidic device for detecting a target gene, comprising:
a plurality of capillaries partially immersed in a sample container containing a sample solution, and the sample solution flows in the plurality of capillaries by a capillary phenomenon, and
a bead filler disposed at one portion in each capillary, the one portion being disposed on a flow path of the sample solution,
wherein each of the bead fillers comprises:
a filler tube disposed at the capillary tube so as to partially constitute the flow path of the sample solution,
a plurality of microbeads contained in the filler tube and closely contacted with each other to form gaps between the microbeads, and
a probe linker formed on a surface of each of the microbeads,
wherein the probe adaptor is configured to amplify a target gene in the sample solution by complementary binding to the target gene, thereby detecting the target gene, and
wherein the void is blocked or reduced in size by amplification of the target gene induced via the complementary binding between the target gene and the probe linker,
wherein a paper that changes color when contacted by the sample solution is disposed at an upper portion of the microbead filler,
whether the sample solution has moved to the upper portion of the capillary is checked by the color change of the paper, so that the target gene can be detected.
2. The microfluidic device for detecting a target gene according to claim 1, wherein an initial volume of the sample solution contained in the sample container is set so that the sample solution reaches the microbead filler through the capillary by a capillary phenomenon,
wherein the void is blocked or the size of the void is reduced by amplification of the target gene induced via the complementary binding between the target gene in the sample solution reaching the bead filler and the probe linker, and
wherein the target gene is detected based on whether the sample solution flows to at least one of an opposite side of the bead filler or a final travel distance of the sample solution when the sample solution is further introduced into the sample container.
3. The microfluidic device for detecting a target gene according to claim 1, wherein the probe linker of each bead filler is configured to detect a different target gene.
4. A microfluidic device for detecting a target gene, comprising:
a sample chamber containing a sample solution;
a microchannel connected to the sample chamber and through which the sample solution flows; and
a bead filler disposed on a flow path of the sample solution in the microchannel;
wherein the microbead filler comprises:
a filler tube disposed at the microchannel so as to partially constitute the flow path of the sample solution,
a plurality of microbeads contained in the filler tube and closely contacted with each other to form gaps between the microbeads, and
a probe linker formed on a surface of each of the microbeads,
wherein the probe adaptor is configured to amplify a target gene in the sample solution by complementary binding to the target gene, thereby detecting the target gene, and
wherein the gap is blocked or reduced in size by amplification of the target gene induced via the complementary binding between the target gene and the probe linker, thereby altering a final travel distance of the sample solution, a time taken for the final travel distance, and a flow rate of the sample solution, and wherein the target gene is detected by one of the final travel distance, the time taken for the final travel distance, and the flow rate.
5. The microfluidic device for detecting a target gene according to claim 4, further comprising a negative pressure chamber disposed on an opposite side of the sample chamber to be in fluid communication with the microchannel, the negative pressure chamber applying a negative pressure from outside to flow the sample solution through the microchannel,
wherein the void is blocked or reduced in size by amplification of the target gene induced via the complementary binding between the target gene and the probe linker, thereby detecting the target gene according to a change in the negative pressure applied by the negative pressure chamber.
6. The microfluidic device for detecting a target gene according to claim 4, wherein the sample chamber, the microchannel, and the bead fillers are provided in plural form to be respectively arranged in parallel, and the beads contained in one of the bead fillers do not have the probe linker.
7. The microfluidic device for detecting a target gene according to claim 4, wherein the sample chamber, the microchannel, and the bead filler are provided in plural form to be respectively arranged in parallel, and the probe linker of each bead filler is configured to detect a different target gene.
8. The microfluidic device for detecting a target gene according to claim 4, wherein the microchannel comprises:
a first flow channel connected to the sample chamber and
a plurality of second flow channels branched from the first flow channels,
wherein the bead filler is provided in plural form to be arranged in each of the second flow channels,
wherein the microbeads contained in each microbead filler have different diameters.
9. The microfluidic device for detecting a target gene according to claim 1 or 4, wherein the diameter of the microbeads is set to have a size in a range of 0.1 to 100 micrometers so that the target gene can pass through the gap, according to the type of the target gene.
10. The microfluidic device for detecting a target gene according to claim 1 or 4, wherein the bead filler includes meshes respectively disposed at both ends of the filler tube to prevent the beads from being lost.
11. The microfluidic device for detecting a target gene according to claim 1 or 4,
wherein the probe linker comprises a coating portion coated on a surface of the microbead, a primer attached to the coating portion, and a template complementarily binding to the primer,
wherein the template includes a first binding portion complementarily binding to the target gene, a second binding portion complementarily binding to the primer, and a third binding portion complementarily forming a dumbbell in the template, and wherein the first binding portion is divided and formed at both ends of the template, and the second binding portion is formed between the divided third binding portions.
12. The microfluidic device for detecting a target gene according to claim 11, wherein the coating portion comprises one or more selected from the group consisting of: 5-hydroxydopamine hydrochlorid, norepinephrine, epinephrine, pyrogallol, dopa (3, 4-dihydroxyphenylalanine), catechin, tannic acid, pyrogallol, catechol, heparin catechol, polyglutame catechol, poly (ethylene glycol) catechol, poly (ethyleneimine) catechol, poly (methyl methacrylate) catechol, hyaluronic acid catechol, polylysine catechol, and polylysine.
13. The microfluidic device for detecting a target gene according to claim 11, wherein the primer comprises one or more selected from the group consisting of: thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, carbonate, HOBt ester, glutaraldehyde, carbamate, imidazole carbamate, maleimide, aziridine, sulfone, vinyl sulfone, hydrazine, phenyl azide compound, benzophenone, anthraquinone, and dienyl, and wherein the end of the primer is modified.
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| CN113652341A (en) * | 2020-07-29 | 2021-11-16 | 哈尔滨工业大学 | Micro-fluidic visual field rapid inspection device based on nucleic acid isothermal amplification reaction blocking flow and detection method thereof |
| KR102579071B1 (en) * | 2021-02-18 | 2023-09-15 | 고려대학교 산학협력단 | Micro-fluidic apparatus for detecting nucleic acid with pore structure |
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| CN114854026B (en) * | 2022-04-29 | 2023-04-11 | 中国科学院兰州化学物理研究所 | Modified chitosan, preparation method thereof, biological lubricant and application thereof |
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Also Published As
| Publication number | Publication date |
|---|---|
| EP3434369A4 (en) | 2019-11-06 |
| KR20170110864A (en) | 2017-10-12 |
| US20190113507A1 (en) | 2019-04-18 |
| WO2017164514A1 (en) | 2017-09-28 |
| KR101799192B1 (en) | 2017-11-17 |
| US11204348B2 (en) | 2021-12-21 |
| CN108883412A (en) | 2018-11-23 |
| JP2019513351A (en) | 2019-05-30 |
| EP3434369A1 (en) | 2019-01-30 |
| JP6719582B2 (en) | 2020-07-08 |
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